Abstract 420: On-target mutations confer resistance to WRN helicase inhibitors in Microsatellite Unstable Cancer Cells

Abstract Werner helicase inhibitors (WRNi) are in clinical development for treating patients with microsatellite-unstable (MSI) tumors characterized by defective DNA mismatch repair. Here, we investigate the impact of cancer cell evolutionary adaptation on WRN pharmacological inhibition with implications for understanding drug selectivity and potential clinical resistance. Coupling genome-wide CRISPR screens with WRN gene knockout, no suppressors of WRN dependency were identified, underscoring WRN’s essential non-redundant function in MSI cells. Pharmacogenomic screens pinpointed modulators of WRNi sensitivity, including SMARCAL1, linking it to WRN-MSI synthetic lethality. Semi-saturation mutagenesis of WRN and prolonged drug treatment identified on-target WRN mutations driving acquired resistance to multiple WRNi in vitro and in vivo. Specific resistance mutations induce cross-resistance to WRN inhibitors, whereas others preserve sensitivity to alternative WRNi. Our work guides next-generation strategies for targeting MSI cancers, enabling cross-resistance studies to evaluate current and novel WRNi efficacy and informing future clinical trial design. Citation Format: Gabriele Picco, Yanhua Rao, Angham Al Saedi, Samantha J. Walker, Shriram Bhosle, Yang Lee, Sara F. Vieira, Gilberto Valdes Garcia, Kieron May, Francesco Sassi, Mamta Sharma, Cansu Dincer, Inigo Barrio Hernandez, Theo Bell, Anastasia Kavasakali, Maria Garcia-Casado, Ray Shenje, Cuthbert Martyr, Edward Brnardic, Nanhua Deng, Hristina Grigorova Dimitrova, Emre Karakoc, Emma Hitch, Katrina McCarten, Zoe Hewitson, Howard Lightfoot, Syd Barthorpe, Michael P. DeMartino, Phil Landis, Emanuel Gonçalves, Andrea Bertotti, Livio Trusolino, Geeta Sharma, Matthew A. Coelho, Jonathan Houseley, Benjamin Schwartz, Mathew J. Garnett. On-target mutations confer resistance to WRN helicase inhibitors in Microsatellite Unstable Cancer Cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 420.